The basics of carrier-free deflection electrophoresis, especially with respect to continuously operated processes, have been described in the literature over 30 years ago. The process is sometimes described under the term FFE (free flow electrophoresis) or more commonly CFE (continuous flow electrophoresis). (K. Hannig: Carrier-free continuous electrophoresis and its application. Anal. Chem. 181, 233 (1961); M. C. Roman and P. R. Brown: Anal. Chem. Free Flow Electrophoresis. 66(N2), 86-94, (1994); R. Braun, H. Wagner and G. Weber: Preparative Free Flow Electrophoresis—a powerful procedure for separating natural substances, GIT Fachzeitschrift für das Laboratorium 39 (1995), 317-322).
Generally, FFE separation procedures are used to separate ions of any molecular weight up to bioparticles. It is here irrelevant whether the sample to be separated is charged itself, or whether the charge came about via the addition or sorption of ions.
Carrier-free deflection electrophoresis has traditionally been used in continuous processes commonly referred to as continuous free-flow electrophoresis (CFFE). This method used in absence of a support matrix such as a gel enables the separation, fractionation, and possible isolation of both soluble and insoluble components. In comparison to other methods enabling isolation of separated sample components, continuous free-flow electrophoresis generally offers three main advantages: (i) the sample is maintained in a liquid medium/in solution which can be directly used for further processing, (ii) the separation may be performed continuously and enables one to obtain as much as hundreds of milligrams or even gram amounts of pure substances per hour and (iii) the separation is gentle and preserves biological activity of the separated components.
The technology of FFE is particularly useful in the separation and fractionation of complex proteins, and is thus applicable to the emerging field of proteomics, which is growing increasingly important in academic and pharmaceutical research as well as the general biotechnology and clinical diagnostic markets. For example, as proteomic research has grown, there has been an increased demand in the improvement of protein separation performance or resolution, especially in terms of resolution process reliability. There has also been demand for a universal front-end separation system and method that occurs prior to a later analysis or further separation/fractionation step.
Improvements to the field of FFE have come about. For example, the process of continuous deflection electrophoresis (or continuous free-flow electrophoresis) has been improved by way of stabilization media and counter-flow media. This is reflected, for example, in U.S. Pat. No. 5,275,706, the disclosure of which is hereby incorporated by reference in its entirety. According to this patent, a counter-flow medium is introduced into the separation space counter to the continuous flow direction of the bulk separation medium and sample that travels between the electrodes. Both media (separation media and counterflow media) are discharged or eluted through fractionation outlets typically into a microtiter plate, resulting in a fractionation process having a low void volume. Additionally, a laminar flow of the media in the region of the fractionation outlets is maintained (i.e., with very low or no turbulence).
Additionally, free-flow deflection electrophoresis has been implemented in a non-continuous or interval process. For example, a process of non-continuous deflection electrophoresis is shown in U.S. Pat. No. 6,328,868, the disclosure of which is hereby incorporated by reference. In this patent, the sample and separation medium are both introduced into an electrophoresis chamber, and then separated using an electrophoresis mode such as zone electrophoresis, isotachophoresis, or isoelectric focusing, and are finally expelled from the chamber through fractionation outlets. Embodiments of the '868 patent describe the separation media and sample movement to be unidirectional, traveling from the inlet end towards the outlet end of the chamber, with an effective voltage applied causing electrophoretic migration to occur while the sample and media are not being fluidically driven from the inlet end towards the outlet end. Examples of embodiments of the '868 patent are shown in FIG. 1.
Both above examples of FFE (i.e., continuous and non-continuous or interval mode) can be used in certain situations, with each experimental goal having factors and requirements or specifics that lend one to prefer one process to the other. Such factors include the choice of sample intended to be separated including required or desired separation time, sample size, separation resolution desired, chamber size, etc. These and other factors influence the mode of separation as well as the apparatus, specific methods, techniques, and compositions to be used. One or more of these above factors may or may not influence which mode of operation (continuous or non-continuous) is chosen when both are available to the user given a certain situation and experimental or separation goal. It should be noted that while using free-flow electrophoresis, both in continuous and non-continuous (or interval) modes of operation, each may have many benefits when compared to other separation or fractionation methodologies and techniques. Nevertheless, improvements are always desired.
Numerous publications describe the physical or electrochemical effects that contribute to the so-called “band widening” of the analytes during separation in continuous free-flow deflection electrophoresis (J. A. Giannovario, R. Griffin, E. L. Gray: A mathematical model of free-flow electrophoresis. Journal of Chromatography, 153, 329-352 (1978); F. G. Boese: Contribution to a mathematical theory of free flow electrophoresis, J. Chromat. 483, 145-170 (1988); K. Hannig and H. G. Heidrich: Free-Flow Electrophoresis, 1990 by GIT Verlag Darmstadt ISBN 3-921956-88-9).
The most important of these effects inherent in continuous FFE are:
1. band widening due to the laminar flow profile;
2. band widening due to thermal convection;
3. band widening due to electrical osmosis;
4. band widening due to electrokinetic effects.
The negative influence of all electrokinetic effects described thus far can be minimized or eliminated by using separation media with suitable ionic constituents with sufficiently high ionic strength, and at the same time not excessively increasing the concentration of the sample.
There are numerous ways to minimize the negative influence of electrical osmosis, e.g., through the selection of a suitable wall material (plastics instead of glass or quartz), or most preferably by adding surface-active chemicals to the separation media that preclude electrical osmosis. This method is referred to as “dynamic coating” in the literature.
The negative influence of thermal convection can be reduced very easily by arranging and operating the electrophoresis chamber horizontally instead of vertically. Additionally, thermal effects can be minimized by appropriate cooling and maintaining the electrophoresis chamber at a constant temperature throughout the separation process.
The negative influence of the laminar flow profile is not observed for continuous isoelectric focusing (IEF) as long as a sufficiently long separation time is selected that also enables the focusing of the analytes, which are transported at the highest linear velocity in the center of the electrophoresis chamber gap.
By contrast, the negative influence is very significant in the case of the electromigration processes. Analytes that migrate near or in the boundary surface to the walls of the electrophoresis chamber pass through the electrophoresis chamber in a considerably longer time than analytes at the center of the electrophoresis chamber gap, and are therefore deflected to a clearly greater extent due to their longer residence time. This effect results in a band widening detectable as a tailing in the direction of electromigration.
Given a continuously executed electromigration process under the boundary conditions of carrier-free electrophoresis, the negative influence of the laminar flow profile cannot be averted for low-molecular analytes. The absolute value of band widening increases as does the migration distance of the analytes. Reducing the diffusion rates for analytes that have their migration impacted by using separation media with increased viscosity also does not help, since this magnifies the unfavorable nature of the laminar flow profile.
In the case of separation of bioparticles, a quantitatively reduced sample feeding to the center of the electrophoresis chamber gap can result in an improved resolution, since the particles cannot get into the area of the electrophoresis chamber walls during a retention time of <10 minutes due to the extremely low diffusion. However, the influence of laminar flow profile can only be minimized in this way by distinctly reducing the sample feeding rate (e.g., to a sample flow rate that is only 0.1% to 0.5% of the flow rate of the separation medium).
Compared to the continuous FFE operating modes known in the art, the interval FFE mode as described in U.S. Pat. No. 6,328,868 is capable of avoiding the negative influence of a laminar flow profile observed in electromigration processes.
However, there remains a need in the art for further improvements of the free-flow electrophoresis methods.